1. Field of the Invention
This invention relates generally to the selection of inhibitors with anti-amyloidogenic activity for the treatment of neurodegenerative disease and more specifically to methods of testing and selection of inhibitory compounds with anti-amyloidogenic properties for the treatment of Parkinson's, Alzheimer's and other neurodegenerative diseases.
2. Background Information
Degenerative disorders of the central nervous system are characterized by progressive, subacute or chronic loss of neurons and their terminals accompanied by reactive alterations, including astrogliosis and microgliosis.
Some neurodegenerative disorders are determined genetically while others occur independent of genetics. In general terms, Alzheimer's disease is the most commonly occurring neurodegenerative disorder, followed by disorders associated with the presence of Lewy bodies, such as Parkinson's disease and the Lewy body variant of Alzheimer's disease. Other less frequently occurring neurodegenerative disorders include Pick's disease, Huntington's disease, Progressive supranuclear palsy and Friedreich's ataxia, to name a few.
Neurodegenerative disorders are characterized by damage to selective neuronal populations that could be followed or preceded by synaptic injury. The mechanisms triggering cell death and synaptic damage in these disorders might be related to the increase of a toxic property and/or loss of neuroprotective capabilities of a specific neuronal cell protein. The disruption of the synaptic connections in the brain results in neuronal dysfunction that, in turn, can lead to characteristic symptoms of dementia and/or motor impairment observed in several neurodegenerative disorders.
The progressive neurodegeneration seen in Parkinson's disease exemplifies the process of synaptic and neuronal loss with concomitant plaque and tangle formation and a decrease in specific neurotransmitters. The disease is generally age related, with 66% of patients with parkinsonian symptoms being between 50 and 69 years of age.
The anatomical hallmark of Parkinson's disease is a loss in pigmentation in a region of the midbrain, known as the substantia nigra. Further, the appearance of abnormal cytoplasmic inclusions, labeled Lewy bodies, is another trademark of the disease. The substantia nigra, translated meaning “black substance”, is an area of the brain rich in dopaminergic neurons and the black pigment, neuromelanin. Loss of neurons from this region in the Parkinson's disease brain leads to a dopamine deficit. The loss of cells is visibly apparent, seen as the loss of black pigment.
In addition to the loss of neuromelanin pigment in the substantia nigra, intracellular accumulations of Lewy bodies are found in many of the neurons of a Parkinson's afflicted brain. Lewy bodies are highly insoluble and difficult to characterize but may contain abnormally phosphorylated neurofilament proteins. Antibodies raised to the protein ubiquitin have been particularly useful in identifying Lewy bodies. While the role of ubiquitin in Lewy body formation is unclear, there is speculation that it may be involved in the cell's attempt to remove abnormal proteins by proteolysis. Since these proteins may be somewhat resistant to proteolysis, the build-up of these proteins leads to the inclusions seen around the neuronal cells.
Emerging evidence from genetic, neuropathological and biochemical studies points to the critical roll non-amyloid β component (NACP)/α-synuclein plays in the amyloidogenesis seen in a number of neurodegenerative diseases. NACP/α-synuclein was previously identified as a precursor of non-A β component (NAC) of Alzheimer's disease amyloid, which was co-purified with Aβ from the Alzheimer's disease brain. Mutations within the precursor NACP/α-synuclein of Alzheimer's disease amyloid have recently been linked to familial Parkinson's disease and this molecule has been found to be a major component of Lewy bodies. This would suggest that abnormal accumulations of NACP/α-synuclein might play a role in the pathogenesis of disorders with Lewy bodies. Research by Spillantini, et al. (Nature, 388: 839-40, 1997), Takeda, et al. (Am. J. Pathol., 152: 367-72, 1998) and Baba, et al. (Am. J. Pathol., 152:879-84, 1998) subsequently showed that neuronal inclusion Lewy bodies and positive neurites, observed in both sporadic Parkinson's disease and dementia with Lewy bodies disease, were strongly immunoreactive with anti-NACP/α-synuclein antibodies, and recognized both the N- and C-terminals of the protein. This would indicate that NACP/α-synuclein is aggregated as a full length molecule into Lewy bodies.
A central question raised by many of these experimental findings concerns the molecular mechanism by which NACP/α-synuclein is aggregated under pathological conditions. Previous biochemical studies in vitro have shown that the aggregation of NACP/α-synuclein was stimulated by a number of amyloidogenic peptides and by specific metals, including aluminum and iron. In addition, NACP/α-synuclein was shown to bind phospholipids, causing a dramatic increase in the α-helicity of it's structure which may, in fact, be a prerequisite for the inhibition of aggregation of NACP/α-synuclein.
While it is understood that the aggregation of NACP/α-synuclein may play a critical role in the pathogenesis of Parkinson's and Lewy body disease the exact mechanism of this process remains unclear. As such, it is important to determine which factors promote abnormal aggregation and fibrillogenesis of this molecule if an effective treatment is ever to be found. The cause of selective degeneration of melanized dopaminergic neurons in the substantia nigra of the Parkinson's brain also remains unsolved. While there is speculation that the excessive production of free radicals during dopamine auto-oxidation into neuromelanin may account for cell cytotoxicity, what makes some neurons more susceptible to damage than others is not completely understood.
Thus, the need remains for a method that will allow for the elucidation of the amyloidogenic process seen in neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, as well as a means for the testing and selection of therapeutic agents that have potential anti-amyloidogenic characteristics. The present invention satisfies these needs and provides related advantages as well.